Polymorphonuclear cells (PMNs) from healthy donors and differentiated HL-60 cells were compared in an opsonophagocytic assay using fluorescent latex beads coated with Streptococcus pneumoniae polysaccharide conjugates. Serum-specific phagocytosis was efficiently mediated by both sources of cells, as measured by flow cytometry, but the mean number of beads ingested per cell was three-to fivefold higher when PMNs were used than when HL-60 cells were used. Nevertheless, differentiated HL-60 cells could be a convenient and standardized source of cells to evaluate the functionality of specific antibodies to vaccine candidates as a coating on fluorescent beads.In the search for new vaccines, investigators often have to select a few candidates among a large number of antigens and formulations. A good way to evaluate the potential of such candidates is to measure the specific immune responses against the different antigens in convalescent-phase or resistant hosts after natural infection with the corresponding pathogen. Besides classical enzyme-linked immunosorbent assay techniques, functional assays, such as opsonophagocytic assays, have been demonstrated to be more relevant to analyze the role of antibodies in protection, in particular in the case of Streptococcus pneumoniae (pneumococcal) or Neisseria meningitidis (meningococcal) infections (1-5). An opsonophagocytic assay using fluorescent beads coated with different antigenic structures has been developed by A. Lehmann and coworkers (1), with polymorphonuclear cells (PMNs) from healthy donors as effector cells; opsonophagocytic activity is measured by flow cytometric analysis, the end points being the percentage of fluorescent cells, the mean number of beads per each phagocyte (designated by M in this study), and the product of these two values, the phagocytosis product (PP) (2, 3). In parallel, the group of G. M. Carlone has developed opsonophagocytic assays using differentiated HL-60 cells. In this assay, live bacteria and, more recently, fluorescently labeled and fixed bacteria were used (4, 5). Opsonophagocytic activity is measured in the former case by viable count, while in the latter case, flow cytometry is used.In the present study, we evaluated the combined use of antigen-coated fluorescent beads as targets and HL-60 cells as phagocytes compared to PMNs from healthy donors. Streptococcus pneumoniae polysaccharides from serotypes 4 and 14 conjugated to tetanus toxoid (TT [Pn4-TT and Pn14-TT, respectively]) were used as antigens. The assay was set up with rabbit positive sera (demonstrated in the department to be opsonic with human cells in a viable opsonophagocytic assay) and negative sera (directed against an irrelevant conjugate) in order to have well-identified negative controls, because it was difficult to obtain and select human sera without specific antipneumococcal antibodies. Nevertheless, several human sera, including a reference serum from Sandoz (Sandoglobuline; Sandoz, Rueil Malmaison, France), were tested in a second step. Rabbit sera heated at ...

Gonadotropin-releasing hormone (GnRH) effects on the lactotroph function have been widely studied, but they probably result from paracrine interactions. No visual data about GnRH receptor in the pituitary are available. In order to identify the GnRH target cells in the pituitary of adult rats, the cellular distribution of rat GnRH receptor mRNA was investigated by electron microscopy, usingin situ hybridization on ultrathin pituitary frozen sections.In situ hybridization was performed using a digoxigenin-labeled oligonucleotide probe revealed by an indirect immunogold reaction. Gonadotropin-releasing hormone receptor mRNA was found in the cytoplasmic matrix, apposed to the endoplasmic reticulum and the nucleus of the gonadotrophs, which were identified by their ultrastructural characteristics, and by the presence of luteinizing hormone (LH) immunoreactivity. It was also found in the lactotrophs, which were revealed by the immunocytological detection of prolactin. No GnRH receptor mRNA was detected in corticotrophs, somatotrophs, thyrotrophs or hepatocytes. This result, without excluding paracrine effects, clearly showed that in addition to the gonadotrophs, the lactototrophs are likely to be direct target cells for the hypothalamic GnRH.